It has been estimated that there may be upwards of 1,000 fatty acid structures in nature. Millar et al., (2000) Trends Plant Sci 5(3):95-101. Many of these fatty acids are synthesized by derivatization of the fatty acids by an array of variants of archetypal desaturases. The first of these variant desaturases to be isolated was the Ricinus oleate hydroxylase from castor endosperm, the enzyme responsible for ricinoleic acid synthesis. van de Loo et al., (1995) Proc. Natl. Acad. Sci. USA 92(15):6743-6747. This was followed by the genes encoding the Vernonia linoleate epoxidase and the Crepis oleate actylenase. Lee et al., (1998) Science 280(5365):915-18. The isolation of these genes led to the notion that their heterologous expression in oil crop plants could facilitate the accumulation of the corresponding unusual fatty acids. Broun et al., (1997) Plant Journal 13:201-10. However, the resulting unusual fatty acid accumulation was invariably lower than that found in the natural source plant from which the gene was isolated. Napier, J. A. (2007) Annu. Rev. Plant Biol. 58:295-319.
The specific activity profiles of variant desaturase enzymes that have been isolated from tissues that accumulate unusual fatty acids are consistent with a role of producing the corresponding unusual fatty acids. However, they exhibit very poor specific activities compared with all stearoyl-ACP desaturases reported to date and have proved ineffective in producing altered fatty acid phenotypes when heterologously expressed. Cahoon et al., (1994) Prog. Lipid Res. 33:155-63. For instance, seed-specific expression of the castor hydroxylase under the control of a strong seed-specific promoter in the model plant Arabidopsis resulted in the accumulation of only about 17% of ricinoleic acid, far short of the about 90% found in castor seed. Broun and Somerville (1997) Plant Physiol. 113:933-42. Similarly disappointing results have been reported for epoxy and acetylenic fatty acids which have been reported to accumulate to 15 and 25% respectively upon heterologous expression of the epoxygenase and acetylenase in Arabidopsis. Lee et al., (1998) Science 280(5365):915-18. In addition to showing poor activities, variant desaturases tended to form insoluble aggregates when purified. Low stability and poor catalytic rates are properties shared by many newly evolved enzymes that arise as gene duplication events in which selection for stability and/or turnover is released, while mutations accumulate that finally result in alteration of function. Govindarajan and Goldstein (1998) Proc. Natl. Acad. Sci. USA 95:5545-49; Goldstein (2001) in Protein Folding, Evolution and Design (Broglia, R. A., Shakhnovich, E. I., and Tiana, G., eds) CXLIV Vols., I.O.S. Press, Amsterdam.
Many potential explanations for low levels of target fatty acid accumulation have been advanced. Napier, J. A. (2007) Annu. Rev. Plant Biol. 58:295-319. Evidence suggests specialized enzymes may play a key role in the incorporation of the unusual fatty acid into triacylglycerols. For instance, the accumulation of laurate in transgenic Brassica napus seeds increased from 50% to 60% upon the coexpression of a coconut lysophosphatidic acid acyltransferase along with the California bay medium chain thioesterase. Knutzon et al., (1999) Plant Physiol. 120(3):739-46. Recently, coexpression of the castor type-2 acyl-coenzyme A:diacylglycerol acyltransferase (RcDGAT2) along with the castor hydroxylase increased the accumulation of ricinoleic acid from about 17% to about 30%. Burgal et al., (2008) Plant Biotechnol. J. 6(8):819-31.
Accumulating high levels of unusual fatty acids in transgenic plants equivalent to those found in naturally occurring species has yet to be reported. As unusual fatty acids are highly desirable in a variety of industries and applications, there is a need for better expression of unusual fatty acids in transgenic plants designed for their production.